1. Field of the Invention
The present invention relates to a protein isolated from Coprinus clastophyllus, especially to a protein isolated from Coprinus clastophyllus and having prolyl oligopeptidase activity, the isolated gene sequence thereof, method for producing and use of the same.
2. Description of the Prior Art
Prolyl oligopeptidase (EC 3.4.21.26), also known as prolyl endopeptidase or post-proline cleaving enzyme, cleaves proline containing polypeptides at the carboxyl side of a proline residue (Polgár, Methods Enzymol. 244:188-200; Polgár, Cell. Mol. Life Sci. 59:349-362).
Prolyl oligopeptidase is widely researched in recently in several application fields. Prolyl oligopeptidase degrades peptides involving memory and learning and thus considered to be connected with amnesia and conditions of degradative memory, including Parkinson's disease. Inhibitants for prolyl oligopeptidase are currently being researched to find therapies thereof (Yoshimoto et al., J. Pharmacobio-Dyn. 10:730-735; Atack et al., Nat. Prod. Res. 19:13-22; Marighetto et al., Learn Mem. 7:159-169; Lee et al., Planta Med. 70:1228-1230; Sorensen et al., Nahrung 48(1):53-56; Atta-ur-Rahman et al., Nat. Prod. Res. 19:13-22; Jarho et al., J. Med. Chem. 48:47772-4782). Researches in other application fields include: using prolyl oligopeptidase as a treatment for celiac disease caused by proline abundant gluten (Piper et al., J. Pharmacol. Exp. Ther. 311:213-219; Marti et al., J. Pharmacol. Exp. Ther. 312:19-26; Matysiak-Budnik et al., Gastroenterol. 129(3):786-796; Pyle et al., Clin. Gastroenterol. Hepatol. 3(7):687-94; Gass et al., Biotechnol. Bioeng. 92(6):674-84); purification and recovery of exogenously expressed peptides (Xiu et al., Biotechnol. Appl. Biochem. 36(Pt2):111-117); and development of a cancer-treating prodrug being less toxic to cells and will be converted by prolyl oligopeptidase to a functional drug (Heinis et al., Biochemistry 43:6293-6303).
Prolyl oligopeptidases found in animals, plants and microbes generally display relatively low activities. Some know prolyl oligopeptidases found in microbes include those originate from: Flavobacterium meningosepticum (having an activity of 0.30 U/ml according to Yoshimoto et al., J. Biol. Chem. 255:4786-4792); lactobacillus casei (having an activity of 0.15 U/g according to 6. Habibi-Najafi et al., J. Dairy Sci. 77:385-392); Propionibacterium freudenreichii (having an activity of 4.3 mU/ml according to Tobiassen et al., J. Dairy Sci. 79:2129-2136); a fermented broth of Agaricus bisporus (having an activity of 0.15 U/ml according to Abdus Sattar et al., J. Biochem. 107:256-261); and Xanthomonas spp. (having an activity of 0.15 U/ml according to Szwajcer-Dey et al., J. Baceteriol. 174:2454-2459).
Enzymatic activity of prolyl oligopeptidase may be increased by genetic engineering methods, specifically, cloning a prolyl oligopeptidase gene into host cells such as E. coli (Escherichia coli) followed by exogenous large-scale expression. Prolyl oligopeptidase originally from Shingomonas capsulata exhibited 7-fold higher activity of 0.2 U/ml in E. coli (Yoshimoto et al., Japanese patent JP10066570). A Flavobacterium meningosepticum prolyl oligopeptidase gene encoded protein expressed in E. coli exhibits maximal activity of 0.7 U/ml (Diefenthal et al., Appl. Microbiol. Biotechnol. 40:90-97). A Flavobacterium meningosepticum prolyl oligopeptidase reconstructed by Uchiyama in E. coli exhibits maximal activity of 8.1 U/ml, which further demonstrates specific activity as high as 124 U/mg after purification (Uchiyama et al., J. Biochem. 128:441-447). An Aeromonas hydrophila prolyl oligopeptidase expressed in E. coli exhibits activity of 1.48 U/ml which is 100 fold higher than expressed in original Aeromonas hydrophila strain and exhibits specific activity up to 8.8 U/mg after purification (Kanatani et al., J. Biochem. 113:790-796). An Aeromonas punctata prolyl oligopeptidase expressed in E. coli has 112 fold higher activity than that expressed in original strain and exhibits specific activity up to 67 U/mg after purification (Li et al., Wei Sheng Wu Xue Bao. 2000 40(3):277-283).
In addition, a Pyrococcus furious prolyl oligopeptidase gene encoded protein expressed in E. coli exhibits specific activity of 232 U′/mg (the activity units being alternatively defined and calculated as 1 U′ being equal to 0.1 OD410 per minute by Harwood et al., J. Bacterol. 179:3613-3618 and different from that in aforementioned literatures) and 4 U/mg (Harwood and Schreier et al., Methods Enzymol. 330:445-454) after purification.
Aforementioned examples demonstrate that purified prolyl oligopeptidases expressed in E. coli have higher activity. However, prolyl oligopeptidases of different origins have preferences for interaction conditions. Optimum conditions enable a prolyl oligopeptidase to display full activity; otherwise, only partial activity may be attained. A highly active prolyl oligopeptidase has more potential for use if its optimum conditions are similar to practical conditions where it is applied. When evaluating the potential of a prolyl oligopeptidase in such aspect, optimum temperature and optimum pH are considered. Furthermore, ranges of optimum conditions are defined by retained activity under optimum generic environmental conditions. For example, heat stability is determined by measuring the ratio of retained activity after heating to full activity.
Each of the aforementioned prolyl oligopeptidases have corresponding optimum conditions. The optimum conditions for Aeromonas hydrophila prolyl oligopeptidase are 30° C. and pH 8.0. When preheated at 42° C. for 30 minutes, 50% activity is retained. The activity of an Aeromonas punctata prolyl oligopeptidase reaches optimum activity at 34° C. and pH 8.4. The optimum pH and temperature for Flavobacterium meningosepticum prolyl oligopeptidase are 7.0 and 40° C.; and its activity will be reduced to 50% when heated to 42° C. for 15 minutes (Yoshimoto et al., J. Biol. Chem. 255:4786-4792). When heated at 60° C. for 1 hour, the activity of a Flavobacterium meningosepticum prolyl oligopeptidase mutated with error-prone PCR mutagenesis drop to 50% under conditions of pH7.0 and 30° C. (Uchiyama et al., J. Biochem. 128:441-447). Of all prolyl oligopeptidases expressed in E. coli, Flavobacterium meningosepticum prolyl oligopeptidase exhibits the highest specific activity and showed the best heat stability after mutagenesis with error-prone PCR. However, since Flavobacterium meningosepticum is a pathogen, safety concerns arise for use, despite other prolyl oligopeptidases exhibiting lower heat-stability.
Nevertheless, to find and isolate a prolyl oligopeptidase corresponding to human usage from various organisms requires much research and experimentation for those generally skilled in the art of the present invention. This difficulty was compounded when the prolyl oligopeptidase found in the first screening of Aspergillus niger was later authenticated to be another serine protease. Though certain basidiomycete was known to have prolyl oligopeptidase, there are no filamentous fungi known to have prolyl oligopeptidase so far. As a result, no prolyl oligopeptidase of fungal original has been expressed in E. coli in large scale.
To overcome the shortcomings of available prolyl oligopeptidases, the present invention provides a protein having prolyl oligopeptidase activity, a nucleic acid encoding thereof and methods for producing and using the same to mitigate or obviate the aforementioned problems.